Treating Tumors Using TTFields Combined with ABT-751

ABSTRACT

Viability of cancer cells (e.g., glioblastoma cells) can be reduced by administering ABT-751 to the cancer cells and applying an alternating electric field with a frequency between 100 and 400 kHz (e.g., 200 kHz) to the cancer cells. Notably, experiments show that the combination of ABT-751 and the alternating electric field produces synergistic results for certain glioblastoma cell lines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application62/741,791, filed Oct. 5, 2018, which is incorporated herein byreference in its entirety.

BACKGROUND

Tumor Treating Fields (TTFields) are an effective anti-neoplastictreatment modality delivered via non-invasive application of lowintensity, intermediate frequency, alternating electric fields. TTFieldsexert directional forces on polar microtubules and interfere with thenormal assembly of the mitotic spindle. Such interference withmicrotubule dynamics results in abnormal spindle formation andsubsequent mitotic arrest or delay. Cells can die while in mitoticarrest or progress to cell division leading to the formation of eithernormal or abnormal aneuploid progeny. The formation of tetraploid cellscan occur either due to mitotic exit through slippage or can occurduring improper cell division. Abnormal daughter cells can die in thesubsequent interphase, can undergo a permanent arrest, or canproliferate through additional mitosis where they will be subjected tofurther TTFields assault. Giladi M. et al. Sci Rep. 2015; 5:18046.

TTFields therapy is delivered using a wearable and portable device(Optune®). The delivery system includes an electric field generator,four adhesive patches (non-invasive, insulated transducer arrays),rechargeable batteries, and a carrying case. The transducer arrays areapplied to the skin and are connected to the device and battery. Thetherapy is designed to be worn for as many hours as possible throughoutthe day and night.

In the preclinical setting, TTFields can be applied in vitro using, forexample, the Inovitro™ TTFields lab bench system. Inovitro™ includes aTTFields generator and base plate containing 8 ceramic dishes per plate.Cells are plated on round cover slips placed inside each dish. TTFieldsare applied using two perpendicular pairs of transducer arrays insulatedby a high dielectric constant ceramic in each dish. The orientation ofthe TTFields in each dish is switched 90° every 1 second.

SUMMARY OF THE INVENTION

Aspects described herein provide methods for treating cancer with acombination of TTFields and ABT-751 (i.e.,N-[2-(4-hydroxyanilino)pyridin-3-yl]-4-methoxybenzenesulfonamide).Notably, the combination of ABT-751 and TTFields provides a synergisticresult for certain types of glioblastoma.

One aspect of the invention is directed to a first method of reducingviability of cancer cells. The first method comprises administeringABT-751 to the cancer cells; and applying an alternating electric fieldto the cancer cells. The alternating electric field has a frequencybetween 100 and 400 kHz.

In some instances of the first method, at least a portion of theapplying step is performed simultaneously with at least a portion of theadministering step.

In some instances of the first method, the ABT-751 comprises apharmaceutically acceptable carrier.

In some instances of the first method, the applying step has a durationof at least 72 hours.

In some instances of the first method, the applying step has a durationof at least 8 hours.

In some instances of the first method, the applying step comprisesapplying the alternating electric field for at least three intervals ofat least 16 hours each, with a break between each of the at least threeintervals during which alternating electric fields are not applied.

In some instances of the first method, the frequency of the alternatingelectric field is 200 kHz.

In some instances of the first method, the ABT-751 is administered tothe cancer cells at a therapeutically effective concentration, and thealternating electric field has a field strength of at least 1 V/cm in atleast some of the cancer cells.

In some instances of the first method, the cancer cells compriseglioblastoma cells.

Another aspect of the invention is directed to a second method ofreducing viability of cancer cells. The second method comprisesadministering a microtubule poison to the cancer cells; and applying analternating electric field to the cancer cells. The alternating electricfield has a frequency between 100 and 400 kHz.

In some instances of the second method, at least a portion of theapplying step is performed simultaneously with at least a portion of theadministering step.

In some instances of the second method, the microtubule poison comprisesa pharmaceutically acceptable carrier.

In some instances of the second method, the applying step has a durationof at least 72 hours.

In some instances of the second method, the applying step has a durationof at least 8 hours.

In some instances of the second method, the applying step comprisesapplying the alternating electric field for at least three intervals ofat least 16 hours each, with a break between each of the at least threeintervals during which alternating electric fields are not applied.

In some instances of the second method, the frequency of the alternatingelectric field is 200 kHz.

In some instances of the second method, the microtubule poison isadministered to the cancer cells at a therapeutically effectiveconcentration, and the alternating electric field has a field strengthof at least 1 V/cm in at least some of the cancer cells.

In some instances of the second method, the cancer cells compriseglioblastoma cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows how the growth of A172 cells is affected by TTFields aloneand in combination with ABT-751.

FIG. 2 shows how colony formation of A172 cells is affected by TTFieldsalone and in combination with ABT-751.

FIG. 3 shows how the growth of U87 cells is affected by TTFields aloneand in combination with ABT-751.

FIG. 4 shows how colony formation of U87 cells is affected by TTFieldsalone and in combination with ABT-751.

FIG. 5 depicts the results of cell cycle analysis by flow cytometry.

FIG. 6 depicts an example system for applying TTFields to a person'sbrain.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “treating” refers to ameliorating, inhibiting, reducing growth,inhibiting metastases, and prescribing medication to do the same.

The term “reducing viability of cancer cells” as used herein, refers toreducing the growth, proliferation, or survival of the cancer cells.

The term “therapeutically effective concentration,” as used herein,refers to a concentration sufficient to achieve its intended purpose(e.g., treatment of cancer, treatment of glioblastoma).

Although the treatment of human malignancies has improved dramatically,the treatment options for glioblastoma (GBM) are still limited. A newtreatment modality for GBM is tumor treating fields (TTFields), which isan anti-neoplastic treatment modality that creates alternating electricfields that are delivered to the tumor by insulated transducer arrays.Efficacy of TTFields is attributed to mitotic spindle disruption, whicheventually leads to improper chromosome segregation and mitoticcatastrophe. A phase 3 clinical trial has demonstrated effectiveness ofTTFields during maintenance treatment with the DNA alkylating agenttemozolomide (TMZ), especially in patients responding well to TMZ. Sincethe DNA damaging effects of TMZ mainly occur during DNA replication, theinventors believe that this synergistic response could be attributed toeffects of TTFields during interphase.

FIG. 1 shows how the growth of A172 cells is affected by TTFields (200kHz; 3.3 V/cm) alone and in combination with ABT-751 over the course of72 hours. For the first two bars, both without (−) and with (+) ABT-751,TTFields were not applied. For the next two bars, both without (−) andwith (+) ABT-751, TTFields were applied for three 16 hour intervals, andeach of those 16 hour intervals was followed by an 8 hour intervalduring which TTFields were not applied. For the next two bars, bothwithout (−) and with (+) ABT-751, TTFields were applied continuously for72 hours.

FIG. 2 shows how colony formation of A172 cells is affected by TTFields(200 kHz; 3.3V/cm) alone and in combination with ABT-751 over the courseof 72 hours. For the first two bars, both without (−) and with (+)ABT-751, TTFields were not applied. For the next two bars, both without(−) and with (+) ABT-751, TTFields were applied for three 16 hourintervals, and each of those 16 hour intervals was followed by an 8 hourinterval during which TTFields were not applied. For the next two bars,both without (−) and with (+) ABT-751, TTFields were appliedcontinuously for 72 hours.

FIG. 3 shows how the growth of U87 cells is affected by TTFields (200kHz; 3.3 V/cm) alone and in combination with ABT-751 over the course of72 hours. For the first two bars, both without (−) and with (+) ABT-751,TTFields were not applied. For the next two bars, both without (−) andwith (+) ABT-751, TTFields were applied for three 16 hour intervals, andeach of those 16 hour intervals was followed by an 8 hour intervalduring which TTFields were not applied. For the next two bars, bothwithout (−) and with (+) ABT-751, TTFields were applied continuously for72 hours.

FIG. 4 shows how colony formation of U87 cells is affected by TTFields(200 kHz; 3.3V/cm) alone and in combination with ABT-751 over the courseof 72 hours. For the first two bars, both without (−) and with (+)ABT-751, TTFields were not applied. For the next two bars, both without(−) and with (+) ABT-751, TTFields were applied for three 16 hourintervals, and each of those 16 hour intervals was followed by an 8 hourinterval during which TTFields were not applied. For the next two bars,both without (−) and with (+) ABT-751, TTFields were appliedcontinuously for 72 hours.

Collectively, FIGS. 1-4 show the following: (a) TTFields inhibit growthof both A172 (FIG. 1) and U87 cells (FIG. 3) and lowers clonogenicsurvival of A172 cells (FIG. 2); (b) TTFields synergizes with themicrotubule poison ABT-751 (1 μM, 8 hours) in A172 (FIGS. 1 and 2), butnot in U87 (FIGS. 3 and 4); and (c) short treatment interruptions ofeight hours (3×16 hours) do not affect treatment response (FIGS. 1-4).Data are mean±SEM; n≥5; Statistical significance was determined byone-way ANOVA; Tukey-corrected for multiple comparisons; *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

Most notably, a synergistic effect between TTFields and ABT-751 wasobserved for the A172 cells.

FIG. 5 depicts the results of cell cycle analysis by flow cytometry.These results reveal that TTFields treatment in S-synchronized A172cells cause a significant accumulation in G2, leading to delayed mitoticentry and, subsequently, lower entry into G1. Data are mean±SEM; n≥4;Statistical significance was determined by testing whether onethird-order polynomial fit adequately fits all data sets;Tukey-corrected for multiple comparisons; *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

In all the experiments described herein, TTFields (200 kHz; 3.3V/cm)were applied using an Inovitro™ system.

The data described above in connection with FIGS. 1-5 elucidate theeffects of TTFields on the cell cycle and design sensitizing strategiesthat can be implemented using TTFields. The data reveals that (1)TTFields effectively delay growth and decrease clonogenic survival ofGBM cells; (2) TTFields synergizes with the microtubule poison ABT-751;and (3) TTFields cause G2 arrest. Without being bound by this theory,this may be through induction of DNA damage during interphase. Notably,equal effectiveness was found in the interrupted treatment schedule(i.e., when TTFields were applied for three 16 hour intervalsinterrupted by 8 hour breaks) as compared to constant treatment (i.e.,when TTFields were applied continuously for 72 hours).

These results establish that the viability of cancer cells can bereduced by administering ABT-751 to the cancer cells and applying analternating electric field with a frequency between 100 and 400 kHz tothe cancer cells.

In the in vitro context, the administering of the ABT-751 to the cancercells occurs continuously from a first time (t₁) when the ABT-751 isintroduced into the container that is holding the cancer cells untilsuch time (t₂) as the ABT-751 is removed or exhausted. As a result, ifTTFields are applied to the cancer cells between t₁ and t₂, the applyingstep will be simultaneous with at least a portion of the administeringstep.

While the in vitro experiments described above were performed using thefrequencies, field intensities, and durations noted above, thoseparameters may be varied. For example, the frequency could be between100 and 400 kHz, the electric field intensity could be between 0.5 and 5V/cm; and the duration could be anything longer than 8 hours. Moreover,while the experiments were performed using a specific microtubule poison(i.e., ABT-751), a different microtubule poison could be substituted forthe ABT-751.

In the in vitro experiments using the Inovitro™ system described herein,the direction of the TTFields was switched at one second intervalsbetween two perpendicular directions. But in alternative embodiments,the direction of the TTFields can be switched at a faster rate (e.g., atintervals between 1 and 1000 ms) or at a slower rate (e.g., at intervalsbetween 1 and 100 seconds).

In the in vitro experiments using the Inovitro™ system described herein,the direction of the TTFields was switched between two perpendiculardirections by applying an AC voltage to two pairs of electrodes that aredisposed 90° apart from each other in 2D space in an alternatingsequence. But in alternative embodiments the direction of the TTFieldsmay be switched between two directions that are not perpendicular byrepositioning the pairs of electrodes, or between three or moredirections (assuming that additional pairs of electrodes are provided).For example, the direction of the TTFields may be switched between threedirections, each of which is determined by the placement of its own pairof electrodes. Optionally, these three pairs of electrodes may bepositioned so that the resulting fields are disposed 90° apart from eachother in 3D space. In other alternative embodiments, the electrodes neednot be arranged in pairs. See, for example, the electrode positioningdescribed in U.S. Pat. No. 7,565,205, which is incorporated herein byreference. In other alternative embodiments, the direction of the fieldremains constant.

In the in vitro experiments using the Inovitro™ system described herein,the electrical field was capacitively coupled into the culture becausethe Inovitro™ system uses conductive electrodes disposed on the outersurface of the dish sidewalls, and the ceramic material of the sidewallsacts as a dielectric. But in alternative embodiments, the electric fieldcould be applied directly to the cells without capacitive coupling(e.g., by modifying the Inovitro™ system configuration so that theconductive electrodes are disposed on the sidewall's inner surfaceinstead of on the sidewall's outer surface).

The methods described herein can also be applied in the in vivo contextby applying the TTFields to a target region of a live subject's body.This may be accomplished, for example, by positioning electrodes on orbelow the subject's skin so that application of an AC voltage betweenselected subsets of those electrodes will impose the TTFields in thetarget region of the subject's body.

FIG. 6 depicts an example system 20 for applying TTFields to a person'sbrain (e.g., to treat glioblastoma). The system 20 includes an ACvoltage generator 30, a first set of electrodes 44 positioned on theright and left side of the head, and a second set of electrodes 42positioned on the front and back of the head. (Because FIG. 6 depictsthe front view of the scalp 40, the electrodes 42 that are positioned onthe back of the head are not visible in this view.) In the illustratedembodiment, each of the electrodes 42, 44 includes nine circularelements that are wired in parallel. But in alternative embodiments, adifferent number of elements and/or elements with different shapes maybe used, depending on the anatomical location where the electrodes willbe positioned.

To use this system, the first set of electrodes 44 is applied to thesubject's body (i.e., on the right and left sides of the head in theillustrated embodiment). The first set of electrodes 44 is positionedwith respect to the target region so that application of an AC voltagebetween the electrodes 44 will impose TTFields with a first orientation(i.e., right to left in the illustrated embodiment) through the targettissue (i.e., the brain in the illustrated embodiment). The second setof electrodes 42 is also applied to the subject's body (i.e., on thefront and back of the head in the illustrated embodiment). The secondset of electrodes is positioned with respect to the target region sothat application of an AC voltage between the electrodes 42 will imposeTTFields with a second orientation through the tissue (i.e., front toback in the illustrated embodiment). The first orientation and thesecond orientation are different (and are roughly perpendicular in theillustrated embodiment).

After the first and second set of electrodes 42, 44 have been applied tothe subject's body, the AC voltage generator 30 repeats the followingsteps in an alternating sequence: (a) applying a first AC voltagebetween the electrodes of the first set 44, such that TTFields with thefirst orientation is imposed through the tissue and (b) applying asecond AC voltage between the electrodes of the second set 42, such thatTTFields with the second orientation is imposed through the tissue.

In some embodiments, all the electrodes are positioned on the subject'sbody (as depicted in FIG. 6); in other embodiments, all the electrodesmay be implanted in the subject's body (e.g., just beneath the subject'sskin, or in the vicinity of the organ being treated); and in otherembodiments, some of the electrodes are positioned on the subject's skinand the rest of the electrodes are implanted in the subject's body.

In some embodiments, the electrodes are capacitively coupled to thesubject's body (e.g., by using electrodes that include a conductiveplate and also have a dielectric layer disposed between the conductiveplate and the subject's body). But in alternative embodiments, thedielectric layer may be omitted, in which case the conductive plateswould make direct contact with the subject's body.

Optionally, thermal sensors (not shown) may be included at theelectrodes, and the AC voltage generator 30 can be configured todecrease the amplitude of the AC voltages that are applied to theelectrodes if the sensed temperature at the electrodes gets too high.

Note that while FIG. 6 depicts an embodiment in which the TTFields areapplied to the brain, the TTFields may be applied to different portionsof a subject's body as described above in alternative embodiments.

In the in vivo context, the administering of the ABT-751 to the subjectmay be performed using any of a variety of approaches including but notlimited to intravenously, orally, subcutaneously, intrathecal,intraventricularly, and intraperitonealy. And the application of theTTFields to the cancer cells may be performed using the Novocure Optune®system or a variant thereof that operates at a different frequency. Inthe in vivo context, the administering of the ABT-751 to the cancercells can occur continuously from a first time (t₁) when the ABT-751 iscirculating in the patient's body (e.g., after administering itsystemically) or introduced into the vicinity of the cancer cells untilsuch time (t₂) as the ABT-751 is eliminated from the patient's body orexhausted. As a result, if TTFields are applied to the cancer cellsbetween t₁ and t₂, the applying step will be simultaneous with at leasta portion of the administering step.

Note that any of the parameters for this in vivo embodiment (e.g.,frequency, field strength, duration, direction-switching rate, and theplacement of the electrodes) may be varied as described above inconnection with the in the vitro embodiments.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the sphere andscope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

What is claimed is:
 1. A method of reducing viability of cancer cells,the method comprising: administering ABT-751 to the cancer cells; andapplying an alternating electric field to the cancer cells, thealternating electric field having a frequency between 100 and 400 kHz.2. The method of claim 1, wherein at least a portion of the applyingstep is performed simultaneously with at least a portion of theadministering step.
 3. The method of claim 1, wherein the ABT-751comprises a pharmaceutically acceptable carrier.
 4. The method of claim1, wherein the applying step has a duration of at least 72 hours.
 5. Themethod of claim 1, wherein the applying step has a duration of at least8 hours.
 6. The method of claim 1, wherein the applying step comprisesapplying the alternating electric field for at least three intervals ofat least 16 hours each, with a break between each of the at least threeintervals during which alternating electric fields are not applied. 7.The method of claim 1, wherein the frequency of the alternating electricfield is 200 kHz.
 8. The method of claim 1, wherein the ABT-751 isadministered to the cancer cells at a therapeutically effectiveconcentration, and wherein the alternating electric field has a fieldstrength of at least 1 V/cm in at least some of the cancer cells.
 9. Themethod of claim 1, wherein the cancer cells comprise glioblastoma cells.